The concept of immunity in dental caries

The concept of immunity in dental caries II. Specific

immune

responses

W. Sims, Ph.D., P.D.S., M.R.C.Path., PATHOLOGY

DEPARTMENT,

ROYAL

DENTAL

London, England HOSPITAL,

UNIVERSITY

OF LONDON

It was to explore the possibility that antibodies might be of value in preventing dental caries that the experiments reported in this article were carried out. Negative results are always disappointing but, in summary, they show that if antibodies specific for cariogenic bacteria are present in saliva at all, they are in very low concentration and, even if the amounts of antibody were greatly increased, there is no evidence that they would in any way modify the progress of dental caries.

M

uch of the recent work on the bacteriology of dental caries strongly suggests that the disease is caused by certain distinct types of streptococci which have the general characteristics of Streptococcus mutans.leS As before, when lactobacilli were thought to be the organisms responsible for dental caries,B-8 the idea of preventing or controlling the disease by immune responses is again in fashion.g-21 That the rationale of studies concerned with immunity to dental caries which were carried out in the lactobacillus era now appear faulty is to be expected, but the reasoning displayed in some recent publications is incredibly nai’ve. They seem to infer that, because serum antibodies are protective in diseases such as diphtheria or tetanus, they must necessarily be of consequence in dental caries. Practically all the work on immunity to bacterial diseases has been concerned with virulent, pathogenic organisms which produce toxins or possess antigenic components which enable them to resist the host’s defenses and produce local or systemic infections. It is irrational to assume that the findings of these studies can be applied to a disease that involves a mixed bacterial conglomerate, applied to the surface of an immunologically unreactive tissue, at a site effectually external to the body. The “cariogenic streptococci” are benign members of the normal oral flora; in this laboratory it is exceedingly rare to isolate them from the common, low-grade, mixed infections which arise in the mouth. Harmless bacteria such as these form the bulk of all normal floras. If, by chance, such organisms gain access to the tissues, they are rapidly destroyed, but this does not preclude them from 69

70

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Oral July,

Surg. 1972

persisting on tissue surfaces, often in enormous numbers, throughout the life of the host. The idea that bacteria in normal floras can be controlled or eliminated by boosting the host’s immune responses to them by means of vaccines is one that has credence only in the narrow world of caries immunity. Nevertheless, the notion that vaccines could prevent dental caries has received considerable publicity and created a general expectation of success. Conceivably, antibodies could combat eariogenic streptococci by facilitating their destruction by phagocytosis or some lytic process or, by coating the bacterial surface, interfere with metabolism or prevent aggregation on the tooth surface. The work reported in this article was intended to examine some of these possibilities. Since immune responses cannot be mounted via enamel, the only way in which immune elements can reach the organisms responsible for dental caries is for them to be liberated into the saliva or, in the case of cells, to migrate over the tooth surface. Clearly, the element of chance in this is huge. To ensure the success of an immune response under these conditions would require large numbers of cells or high concentrations of antibody; neither condition obtains in the mouth.22l 23 Although sensitized lymphocytes may be present in the mouth, their numbers must be very small, and the possibility of cell-mediated immune responses being involved in dental caries is so remote that it will not be considered further. Only antibodies of the IgG and IgA types occur in measurable quantities in saliva. The level of IgG antibody in saliva rises and falls with the albumin level, and it now seems established that the amount of IgG antibody in saliva depends on the degree of “leakage” of serum through the oral tissues.24-29 The gingival crevice is a principal route by which serum enters the mouth; the flow is negligible when the supporting tissues of the teeth are healthy, but it increases markedly when they are inflamed.26l 3o Obviously, the amount of IgG antibody in saliva is highly variable; a reasonable average value would be 1.0 mg. per 100 ml., but it may be several times higher than this or it may be present only in trace amounts. When the level is exceptionally high, other antibody types, such as IgM, become detectable. The situation, then, is that IgG antibody is found in saliva in only very small amounts, and, because it is in fact a high dilution of serum antibody, only a tiny fraction of this very small quantity could be expected to consist of antibodies which react specifically with antigens on cariogenic streptococci. Since the amount of IgG antibody in saliva is related to the extent of soft-tissue inflammation in the mouth and not the degree of caries activity, it appears exceedingly unlikely that the IgG antibodies represent an immune response to the organisms causing dental caries. The principal antibodies in saliva, as in other mucous secretions, are of the 11s IgA variety. There are several excellent recent reviews of this type of antibody.31-33 Secretory IgA antibody is produced locally by plasma cells in the salivary glands and the submucous tissues.23l 27*31-3g These plasma cells apparently secrete normal serum type 7s IgA antibody; however, before the antibody reaches the saliva, pairs of molecules become bound together by means of a small glycopeptide known as the “secretory piece” which is produced by the epithelial cells, and this dimeric form is the 11s IgA secretory type antibody. The only special properties possessed by this type of antibody are a readiness to form complexes with other proteins and a greater resistance to digestive enzymes.31-33

Volume 34 Number 1

Immunity

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71

Since IgA antibody is locally produced in response to local antigenic stimulation, the amounts in serum and saliva are unrelated. 2al 21*g1-38This means that it is impossible to make any sensible assertions regarding the studies in which the level of serum antibodies specific for lactobacilli or streptococci were compared with the degree of dental caries. In all the studies concerned with vaccines for dental caries, the vaccine was administered parenterally. This procedure ought to raise the level of serum antibodies, but the only way in which the antibody concentration in saliva could be affected is by the unpredictable “leakage” of serum into saliva already discussed. Estimations of the concentration of IgA in saliva have usually been carried out by concentrating the saliva and then measuring the antibody level by the radial diffusion method of Mancini, Carbonara, and Heremans.‘O Most workers, including myself, have been obliged to use a standardized serum for comparison in this technique. The correct standard to use is, of course, a solution containing a known concentration of 11s IgA secretory antibody which has a molecular weight of 390,000 as contrasted with 170,000 for the serum 7s IgA. To correct for the lesser diffusion of the larger 11s molecule, the results obtained by the radial diffusion method using a serum standard must be increased by a factor of and an 11s approximately three. 32 Using the technique of electroimmunodiffusion IgA secretory antibody standard, Claman, Merrill, and Hartley23 found the concentration of IgA in unconcentrated parotid fluid to be within the range of 2.3 to 15 mg. per 100 ml. with a median value of 9.5 mg. The concentrations in mixed submandibular and sublingual fluid were only slightly more than one half of the parotid fluid values. Despite the problems in estimation, it is clear that secretory antibody is present in saliva only in small amounts. It is being continuously secreted, however, and it is conceivable that an organism attached to the tooth surface, by being continuously bathed in this dilute solution of antibody, could adsorb an effective amount. On the other hand, when one deducts from this small quantity of antibody that portion which has been shown to react with organisms other than cariogenic streptococci,24* 25*sb *Id4 and allowance is made for the fact that cariogenic streptococci, like the lactobacilli, are serologically heterogenous, 46-48it seems highly improbable. A further point is that cariogenic streptococci are found almost exclusively in plaques on tooth surfaces I* 4* 48 but the salivary IgA antibody concentration reaches “adult” levels only ‘s weeks after birth, 27long before the teeth erupt. Polymorphonuclear leukocytes migrate through the oral mucosa into the mouth.50* 51 Most enter through the gingival crevices, and the number migrating is substantially increased when the periodontal tissues are inflamed.52-54 The recent demonstration that the number of polymorphs in saliva varies markedly from person to person and shows large fluctuations in the same individual at different times during the dayZ2 casts doubt on the finding that caries-free persons have more polymorphs in their saliva than those who are caries active.55 Oral polymorphs remain functional only while they remain within the layer of mucous immediately adherent to the mucosa; they degenerate rapidly on escaping into the saliva.50p 51 Since for every degenerating polymorph in saliva there are at least 10,000 bacteria, the idea that the function of these cells is to regulate the oral flora is manifestly absurd. In the totally different conditions prevailing

72

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surg. 1972

in the tissues, a polymorph lives only a Sew honrs; when in motion, it moves randomly at a speed of 40 microns per ini~~1~tc, and the maximum range over which it may be chcmotaciically attractutl js 100 n~icrons.5o 7‘0 ha\-c any effect in dental caries, a polymorph ~vonltl have to Icavc the soft tissues and tllen, keeping strictly within the iJi~~(w~is layer irnmediatcl~- atlhefcnt to the teeth, coniplete a prodigious journ~~y to i;hc tlcntal plaque. JCn route tlw ~11 would em counter

enough

bacteria

to

satisfy

its

~~llagoq-tic

capacity

iJu~uJiie~alde

tirncs

over. On reaching the dental plaqur~, tlic> pol~rno~ph would bc confrontecl with an almost solicl mass of bil(+(~ria antI li?-tlrog~9 and fluoride ion collccllt~ri~tionS that would render it instantly fun&ionlr>ss. ‘I’lx utter improbabilit>~ of oral polymorphs constituting a d&nsc nieclmnism in tlcnt.al caries is obvious. Tliest: cells almost certainly rcprtx~nt t,lic cud rrsull, of the host’s defcnscs against bactcria or other foreign particles whirl1 IIM~ tmwtratrt irrt,o the snperfirial layer of the oral mucosa, so that when the polynrorph emer*gcs into the oral eavi1.y its task is, in fact, complett~tl. If the polpmorphs in saliva arc not ~~onecr~~cl in dcrltitl earit~s, it hc~comes very difficult. to postulate :I rolt3 for ilJltilKJtliW. Of the few ways ill which salivary antibodies might conceivably afford protcc~tion from , saliva slxxinlc~ns I’roln two groups of dental students who differed markedly in caries cspcricnce were csamined for the presence of antibodies to cariogenic strcl)tococei. The indirtct fluorescent antibody tech. nique was used bccausc, by use of :I single fluorcsccnt reagent, antibodies to ;I multiplicity of antigens ci11l be detcrtetl. Rabbit antibody was prepared against. two strains of cariogenic strcptococei and an oral lactobacillus and used in tests involving phagocytosis of these organisms anct t,hcir acid-producing capability. MATERIALS Estimation

AND of

METHODS

immunoglobulin

levels

in

saliva

Saliva specimens wore c~ollected f’rom 1wo groups of twelve dental students with contrasting high am1 low caries cspcrience. Each 20 ml. specimen was centrifuged to deposit partkuhik ninttc>r, alld thcri the clear Sllp~‘~~Jl~~t~:lJlt TV;IS

Volume 34

Number

Immunity

1

in dental caries 73

concentrated approximately twenty times by dialysis against polyethylene glycol for 24 hours. The levels of IgG and IgA antibodies in the concentrated saliva specimens were estimated by the method of radial diffusion using Behringwerke “Partigen” immunodiffusion plates and a standardized reference serum obtained from Hoechst Pharmaceuticals, Ltd. The immunoglobulin levels in the pooled saliva specimensof each group and in a pool of all specimenswere also measured. A graph was constructed for each plate by plotting the squares of the diameters of the circles of precipitate produced by the standard serum dilutions, and the values for the saliva specimens were read off. Examination

of saliva

specimens

for antibodies

specific

for cariogenic

bacteria

The bacteria employed included eight strains of cariogenic streptococcistrains FA-1, GS-5, H&6, Ingbritt, OMZ-51, and OMZ-52, which were kindly supplied by Dr. Diana E. R. Cornick, and two strains of Streptococcus mutans isolated by me: strain NCTC 10449 from deep carious dentine and strain Sm121 from saliva. These strains and an oral strain of Str. sanguis were utilized after growth on blood agar and on blood agar containing 5 per cent sucrose. Oral strains of Lactobacillus casei, L. casei var. rhamnosus, L. acidophilus, L. fermenti, St. hominis (salivarius), and Str. pyogenes (NCTC 8198) were used after growth on blood agar only. Smears were prepared from 24-hour subcultures of these organisms grown anaerobically at 37O C. in an atmosphere of hydrogen containing 5 per cent carbon dioxide. For each saliva specimen, two sets of four slides with six well-separated circles of 5 mm. diameter inscribed on each slide were prepared. Smears of the organisms in phosphate-buffered saline solution (PBS), pH 7.2, were made in the circles. The slides were fixed for 1 minute in 95 per cent ethanol and then washed in PBS. Blobs of the concentrated salivas were applied to the smears, and the slides were placed in a moisture box at room temperature for 1 hour. The slides were then thoroughly washed in PBS and allowed to dry, and then drops of anti-human IgG serum conjugated with fluorescein isothiocyanate were applied to one set of slides and the corresponding IgA antiserum to the other. These sera were obtained from Hoechst Pharmaceuticals, Ltd. The fluorescein: protein ratios, which were determined by measuring the absorption at 495 nm. and 280 nm., were 0.94 for the IgG serum and 0.90 for the IgA. The slides were replaced in the moisture boxes for 30 minutes and then washed thoroughly and allowed to dry. Negative controls consisted of slides treated with saline solution instead of saliva. The slides were examined with a Zeiss Universal fluorescence microscope using incident illumination for excitation of fluorescence and appropriate filters. A test result was considered positive when the bacteria acquired a distinct, complete outer rim of fluorescence clearly distinguishable from the weak, irregular fluorescence occasionally exhibited by the negative controls. Since there was no positive control, a check on the technique was carried out. Rabbit antisera prepared against strain Ingbritt, Str. mutans and L. oasei (see below) were diluted and tested by the indirect fluorescent antibody technique, using a fluorescein-conjugated antirabbit immunoglobulin antiserum (Wellcome Reagents, Ltd.) to determine the highest dilutions which still gave strong posi-

74

Oral July,

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Surg. 1972

tive fluorescence when applied to smears of their specific bacteria. The dilutions were found to be l/300 for strain Ingbritt and Str. mutans and l/500 for L. casei. One milliliter of each of these dilutions of antisera was added to 17 ml. of saliva, which was then thoroughly mixed, centrifuged, and concentrated as described above. There was no discernible difference between the intensity of fluorescence obtained when this concentrated serum-saliva was applied to smears of the three organisms in the indirect fluorescent antibody method and that obtained with the dilutions of antisera. No specific Auorescence was detectable when concentrated saliva without added antisera was used in the test. Effect

of antibodies

on acid

production

Vaccines of strain Ingbritt, Str. mutans (NCTC 10449), and L. casei were prepared by heavily inoculating blood agar plates, which were then incubated for 18 hours at 37O C., and harvesting the growth into sterile saline solution. The cells were washed three times in sterile saline solution and finally resuspended to give a concentration equivalent to Brown’s opacity tube No. 4. The vaccines were heated in the water bath at 60° C. for 30 minutes. Before the injections in rabbits were commenced, serum was obtained from each animal and absorbed with packed, twice-washed cells of the appropriate organism. Four volumes of serum were absorbed with one volume of packed cells for 2 hours at 37’ C. These absorbed sera were used as normal control sera in the tests. Rabbits were injected intravenously with 1.0 ml. of vaccine every 3 or 4 days for 3 weeks. At the end of this time, sera from trial bleedings gave agglutination titers of l/640 for strain Ingbritt, l/1280 for Str. mutans, and l/5120 for L. casei. The rabbits were bled from the marginal ear vein, and the blood was allowed to clot at room temperature. The clots were rimmed and the sera were allowed to separate in the refrigerator overnight. The sera were decanted, clarified by centrifugation, dispensed in aliquots of 2 ml., and stored frozen at -20° C. until required. Suspensions of the three organisms approximately equivalent to Brown’s opacity tube No. 5 were prepared as described for vaccines. The suspensionswere spun down and resuspended in one tenth of the original volume. For each test, 1.0 ml. of this suspension was transferred to a sterile 10 mm. by 50 mm. flatbottomed tube, and the organisms were deposited by centrifugation. The bacteria were then resuspended in one of the following: 1.0 ml. of sterile saline solution, 1.0 ml. of normal rabbit serum, 0.7 ml. of immune rabbit serum + 0.3 ml. of sterile saline solution, 0.7 ml. of immune serum + 0.2 ml. of lyophilized guinea pig complement (Flow Laboratories) freshly reconstituted in sterile saline solution + 0.1 ml. of sterile saline solution or 0.7 ml. of immune serum + 0.2 ml. of complement + 0.1 ml. of a 500 pg per milliliter sterile saline solution of lysozyme (British Drug Houses). After incubation in the water bath at 37O C. for 1 hour, the bacteria were spun down, washed once in 2.0 ml. of saline solution, and finally redeposited to form a flat disk, or “plaque,” of bacteria on the base of the tube. A loopful of the final saline suspension was transferred to a glass slide, fixed, and treated with fluorescein-conjugated anti-rabbit gamma globulin serum as previously described. The tube was placed in a specially drilled-out aluminum alloy block in a constant-temperature “dri-block” heater unit (Techne, Ltd.) and

Volume 34 Number 1

Imnaunity

Sterile Buffer 10% solution sucmse solution -

in dental

caries

75

electrode

Thermometer Constant temperature aluminium alloy block layer of bacteria [‘plaque’ I

Fig. 1. A diagram of the apparatus used to measure the rates of acid production films, or “plaques,” of bacteria when moistened with a solution of sucrose.

of dense

5 minutes was allowed for the temperature to equilibrate at 37O C. Next, a “micro” combined reference and glass electrode ( Pye-Ingold, Ltd.) for measuring the pH of moist surfaces was lowered almost in contact with the bacteria. A Terumo microsyringe (Shandon Scientific Co., Ltd.), the 100 mm. needle of which just passedbetween the side of the electrode and the wall of the tube, was used to apply 0.02 ml. of sterile 10 per cent sucrose solution to the bacterial mass. The electrode was instantly eased into contact with the bacteria and the subsequent pH change with time was recorded. Fig. 1 shows a diagram of the apparatus. During preliminary testing of the technique it was established that saline suspensions of the three organisms could be kept at room temperature for at least 8 hours without any detectable falling-off in acid-producing capacity. All the experiments were completed well within this time period. Phagocytosis

of cariogenic

bacteria

The technique employed was a slightly modified version of that described by Hirsch and Strauss61 Polymorphs were obtained from the peritoneal cavity of the rabbit by injecting 200 ml. of saline solution containing 1.0 mg. ml. of glycogen and then, 4 hours later, washing out the cavity with 200 ml. of saline solu-

76

Sims

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Burg. 1972

tion containing 40 pg per milliliter of heparin. The abdominal wall was gently kneaded, and the exudate was aspirated with a No. 1 serum needle and a 100 ml. syringe. The cells were washed twice in heparinized saline solution and then resuspended in test medium. The test medium was Hank’s solution (Oxoid, Ltd.) containing 0.005 M barbital buffer (pII 7.3), but without added bicarbonate or gelatin. An improved Neubauer counting chamber was used to estimate the number of polymorphs in the suspension, which was then diluted in test medium to give a final concentration of approximately 20 x 10e polymorphs per milliliter. The exudates always consisted of polymorphs in excess of 95 per cent, and no attempt was made to remove the other cell types. Suspensions of strain Ingbritt, Xtr. ntutam, and L. casei were prepared as described for vaccines except that the suspensions were made up equivalent to Brown’s opacity tube No. 1. Previous work had shown that diluting the strain Ingbritt suspension l/160, the Str. mutans suspension l/180, and the L. casei suspension l/130 gave suspensions containing approximately 4 x lo6 viable organisms per milliliter. These dilutions were prepared in test medium. The rabbit’antisera were diluted in test medium to an agglutination titer of l/200.

Tests were carried out in sterile 15 mm. x 50 mm. screw-capped plastic vials; each test consisted of two test vials, set up in duplicate, and three control vials. The test vials contained 1.0 ml. of polymorph suspension and 0.5 ml. of bacterial suspension, with 0.5 ml. of the appropriate antiserum added to one vial and 0.5 ml. of plain test medium to the other. The first control vial contained 0.5 ml. of bacterial suspension plus 1.5 ml. of test medium; the second contained 0.5 ml. of bacterial suspension plus 0.5 ml. of antiserum plus 1.0 ml. of test medium; and the third contained 0.5 ml. of bacterial suspension plus 1.0 ml. of polymorph suspension plus 0.5 ml. of test medium. This third control was gently mixed and stood in the incubator at 37O C. The remaining six vials were clipped to a revolving mixer in the incubator and tumbled end over end thirty-seven times per minute. After incubation for 1 hour, 0.5 ml. amounts from each vial were serially diluted l/10, l/100, and l/1000, and 0.1 ml. amounts from each dilution were spread over the surface of two plates of medium. Blood agar plates were used for counting strain Ingbritt and Str. mutans, and MRS medium (Difco) was used for counting L. casei. Colonies were counted after incubation at 37O C. for 72 hours. Because of the results obtained in the foregoing experiments, similar tests using strains FA-1, GS-5, L. fermenti, and St. pyogenes were carried out. Suspensions of these organisms were prepared from growth in 0.5 per cent glucose digest broth using the previously obtained knowledge of the numbers reached in this medium after incubation for 18 hours at 37O C. The tests involving antisera were omitted; otherwise, the procedure was as described above. Bacteria

in oral

polymorphs

Each subject allowed saliva to collect in the mouth for 1 minute and then delivered it into a sterile plastic vial. The saliva was mixed, and thick smeara were prepared at once on glass slides. While the smears were still wet, absolute

Volume Number

Immunity

34 1

in dental

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77

I. Concentrations of IgG and IgA immunoglobulins in the individual and pooled salivas of two groups of dental students with contrasting high and low caries experience Table

Low

group

High

IgG w./ 100 ml. saliva

b-4 w./ 100 ml. saliva*

Subject

DMF teeth

it5 A: L: P. P.

5 1 0 0

2.05 1.14 1.63 2.21

0.87 1.52 0.82 1.39

ii 0

0.71 0.89 0.48

4.12 0.42 0.67

ii.:. 2.

; ii

1.85 1.75 1.24 1.62

2.17 1.92 1.40 5.00

S: J:

5

0.75

Mean

2.6

S.D. Pooled saliva, low caries group Pooled saliva, both groups by

caries

*These a factor

DMF teeth

Subject

caries

group

IgG w./ 100 ml. saliva,

‘go

“,“;”

saliva*

;; 23

2.12 0.99 1.45 1.29

0.91 1.28 0.96 3.68

26 El

1.02 0.75 1.02

0.64 2.08 3.70

D. W. vi. s. E

24 ii25

0.83 1.85 0.69 0.57

0.90 1.02 1.93 1.47

2.01

J. W.

25

0.17

4.69

1.36

1.86

Mean

24.7

0.571

1.388

S.D.

-

1.39

1.75

-

1.24

2.01

26 F-2 F: +.

Pooled saliva high caries group

values were obtained with a 78 serum IgA of approximately 3 to allow for the lesser

standard. diffusion

1.06

1.94

0.546

1.349

1.03

2.18

1.24

2.01

They should be increased of 11s secretory IgA.

alcohol was applied and allowed to react for at least 10 minutes. The slides were washed and stained by the Papanicolaou method. Provided the smears were not allowed to dry at any stage, the polymorphs appeared intact and any bacteria within them could be clearly seen. Fifty specimenswere collected from staff, students, and patients visiting the department, and 300 polymorphs in each specimen were examined for the presence of bacteria. RESULTS lmmunoglobulins

in saliva

Table I shows that all the subjects in the study had IgG and IgA type immunoglobulins in their salivas at concentrations similar to those reported by other workers.12p23*31*32 Because a serum 7s IgA standard was used in the estimations, instead of the correct but unavailable secretory 11s IgA, the concentrations of IgA shown in the table should be increased by a factor of approximately three.32 Neither the mean concentrations of IgG for each group nor the mean concentrations of IgA differ significantly when tested at the 5 per cent level. Except for someweak reactions with Str. pyogenes, the tests for the presence of salivary IgG or IgA antibodies specific for oral streptococci and lactobacilli

78

Oral July,

Sims

Surg. 1972

Table II. The rates of acid production recorded when dense aggregates of the bacteria normal rabbit serum, specific immune rabbit serum, immune serum and guinea pig Strewbcocous

Saltie

Normal serum

Antiserum

o-00 O-04 O-18 o-39 O-46 o-54 l-03 l-15

o-00 O-06 O-12 o-31 O-40 O-52 l-05 l-20

o-00 O-03 o-14 O-32 o-37 o-43 O-50 o-59

iii 419 4.8

l-40 1-28 l-58

1-36 l-58 2-18

2: 415 4.4 4.3 4.2 9.:

2-20 2-51 3-33 4-21 5-54 7-38 12-35 9-13

2-43 3-09 3-40 4-16 5-15 6-50 17-12 11-31

PH i:: iii 514 it;

strain

Inabritt

Antiserzc.m + complement

Antiserzlm + complement Normal 1ysoiyme

Saline

o-00 O-03 O-07 O-28 O-38 O-46 l-00 l-13

o-00 O-08 o-22 o-45 O-52 o-59 l-07 1-14

o-00 O-03 o-11 o-41 o-55 l-10 l-32 2-03

O-00 O-02 O-08 O-30 o-39 o-55 l-18 l-50

l-08 l-20 l-32

1-46 1-27 2-03

l-27 l-38 1-53

2-48 3-53 4-56

2-33 3-30 4-36

2-11 l-45 2-50 3-53 5-48 16-07

2-24 2-45 3-11 3-46 4-40 6-05 14-32 9-09

2-10 2-32 3-06 3-50 5-00 7-23 12-56

5-44 6-49 7-56 IO-17 18-02

5-48 7-02 8-31 11-15 -

4.19

3.99

4.02

4.30

4.31

.Si?T~

3:9 3.8 pH at 20 minutes The times

3.98 listed

3.99 are in minutes

and

seconds.

were uniformly negative. No specific fluorescence could be detected when smears of cariogenic streptococci and St. sang&s prepared from growth on blood agar or on blood agar containing sucrose were used. The intensity of fluorescence obtained with the smears of Str. pyogenes was insufficient to be confidently graded as positive but was indubitably greater than the controls. There were eleven salivas which reacted with Str. pyogenes; four were from the low caries group and included one specimen which contained specific IgG and IgA antibodies, one which contained only IgG antibodies, and two with only IgA antibodies. Of the seven reactive salivas from the high caries group, two contained specific IgG and IgA antibodies, two contained IgG antibodies only, and three contained only IgA antibodies. Effect

of antibodies

on acid

production

Table II shows that, when allowance is made for experimental error, the exposure of suspensions of streptococci and lactobacilli to immune sera has little effect on their rates of acid production. The experiments were repeated a number of times, but in no case did the results show any more substantial differences than those listed in the table. The rates of acid production are high and, inevitably, this means that the test system is somewhat crude and probably insensitive to minor changes, but, after all, this is the way acid is produced in the dental

Volume 34 Number 1

Immunity

in dental caries

listed were moistened with sucrose solution after being suspended variously complement, or immune serum + complement + lysozyme Strevtowooua

Anttierum o-00

O-07 O-20 O-46 o-54 l-03 l-18 l-37 2-10 2-45 3-49 4-58 6-47 12-09 19-14

4.40

mutam Antiserwm + wmplenzent o-00 O-01

o-13 o-44 O-56 l-08 l-24 l-42 2-04 2-37 3-18 4-12 5-29 7-20 13-08

4.32

Ladobacillud Antiserum + wmplement 1y.vo.&me o-00

O-03 O-08 o-22 O-27 o-33 o-43 o-57 l-18 2-10 3-21 6-32 9.00 14-07

4.43

Saline o-00

o-21 O-40 l-00 l-07 1-12 l-18 l-27 l-38 l-52 2-69 2-33 3-02 3-38 4-19 5-07 6-05 7-18 a-34 lo-56 16-27 3.78

Normal serum o-00

O-04 o-13 o-33 O-40 o-47 o-55 l-03 1-12 l-28 1-47 2-10 2-40 3-10 3-51 4-31 5-32 6-35 7-46 9-25 3.82

Antiseruln o-00

o-11 O-27 O-50 O-56 l-05 1-12 l-20 l-30 1-41 l-53 2-08 2-26 2-44 3-07 3-32 4-11 5-09 6-40 9-00 14-30 3.80

79

in saline,

casei Antiserum

Ant&m&m + Gmnplemmt

comp;ment o-00

1ysoiyme o-00

o-14 O-42 O-58 l-04 l-09 1-14 l-24 1-25 1-31 l-39 l-48 l-59 2-12 2-28 2-44 3-00 3-20 4-38 7-45 E-02 3.79

O-20 O-48 1-18 1-27 1-35 l-43 l-52 2-00 2-10 2-21 2-33 2-47 3-02 3-20 3-42 4-06 4-35 5-13 6-15 9-49 3.77

plaque. A curious feature of the tests was that an initially high rate of acid production was often associated with a high terminal pH, whereas comparatively slow rates of acid production persisted to produce a low final pH value. In Table II the former effect seems to be a characteristic of the tests involving serum while the latter effect is associated with the saline controls, but in other experiments both effects were recorded with either type of test. All the bacteria in the tests involving immune sera fluoresced brightly when tested by the indirect fluorescent antibody technique, confirming that they were coated with antibody. Effect

of antibodies

on phagocytosis

The results listed in Table III are most interesting in that they show that the bulk of all three bacteria were taken up by the polymorphs in the complete absence of serum. Moreover, the presence of specific antibodies only marginally increased the phagocytosis of strain Ingbritt and made no difference at all in the cme of Str. mutans. Antibody did effect a 6 per cent increase in the uptake of L. carei, and the difference is just significant when tested at the 5 per cent level. Phagocytosis of the other organisms tested without antisera was 96.2 per cent for strain FA-1; 98.1 per cent for strain GS-5; 93.8 per cent for L. ferm-enti; and, in striking contrast, 19.4 per cent for Str. pyogenes. This greatly reduced

80

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III. Results of phagocytosis tests using approximately

lo7 rabbit polymorphs cozcnts x 10s

Control Test Test

Streptococcus strain Ingbritt

me&m

only

Plate count 1 146 Plate count 2 131 Mean count 139 Combined mean count Number of bacteria removed Streptococcus mutans Plate count 1 232 Plate count 2 219 Mean count 226 Combined mean count Number of bacteria removed Lactobacil1U.s oasei Plate count 1 171 Plate count 2 198 Mean count 185 Combined mean count Number of bacteria removed The test vials were incubated at 37” C. for 1 hour and, with the exception

me&ma

195 177 186 203 143 179 161 171 of the control

uptake of Xtr. pyogenes, the only pathogen among the organisms tested, is surely significant. Bacteria

in oral

+

antissrzMn 111 149 130 129

polymorphs

A total of 15,000 polymorphs (300 from each of fifty subjects) were examined, and 880 (5.9 per cent) were found to contain bacteria. Most cells contained one, two, or three bacteria, and the highest number seen in a single polymorph was five. It was, of course, impossible to judge whether the bacteria were actually within the polymorphs or whether the polymorphs without bacteria had, in fact, taken up bacteria and digested them. DISCUSSION

The concentrations of IgG and IgA antibodies in the salivas of the two groups studied were within normal limits and were not related to dental caries experience. Indeed, even though the groups were small, the differences between the mean concentrations were so slight that there is no indication that a study of large groups would be expected to produce a different result. Some workers have interpreted their results as indicating a relationship between salivary immunoglobulin level and dental cariesI2 but, as Tomasi and Bienenstocks2 concluded in their review, the evidence is far from convincing. Data on the sensitivity of the indirect fluorescent antibody technique in comparison with other serologic tests are lacking. With the rabbit sera used in this work, it was found that the agglutination test was between two and ten times more sensitive. The agglutination technique requires a minimum of 0.625 pg per milliliter of antibody,58 and thus approximately 7.0 pg per milliliter of antibody was probably the smallest amount which would give a positive result in the fluorescent antibody tests. Since the concentrated salivas ought to have contained, on the average, 1,450 pg per milliliter (7.25/100 x 20 x 1,000) of anti-

vial

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per test Test

Duplicate phagocytosis tests Counts of szlrvtiing bacteria

medium + polymorphs (not rotated) 106

PolymoTphs 383 x 102

129 118

indicated,

416 x 102 400 x 102 409 1,249,1000

only 431 x 102 403 x 102 417 x 102

x

102

(96.8%)

Polymorphs

+ antiserzlm

284 x 102 308 x 102 233 x 102 342 x 102 259 x 102 325 x 102 292 x 102 1,260,8000 (97.7%)

185 208 197

394 x 102 362 x 102 430 x 102 389 x 102 412 x 102 376 x 102 394 x 102 1,990,600 (98.1%)

426 x 102 437 x 102 432 x 102 425 1,987,500

182 154 168

132 x 103 158 x 103 145 x 103 137 x 1,573,ooo

346 x 102 376 x 102 361 x 102 350 x 1,675,OOO

were

rotated

end over

end 37 times

121 x 103 137 x 103 129 x 103 103

(92.0%) per minute.

404 x 102 429 x 102 417 x 102 x 102 (97.9%) 329 x 102 348 x 102 339 x 102 102 (98.0%)

body only approximately 0.5 per cent of it would have needed to be reactive with antigens on the organisms tested to give a positive result. It seems, then, that if antibodies to cariogenic bacteria were present in saliva their concentration was less than 0.035 pg per milliliter. The failure to detect antibodies reactive with cariogenic bacteria in saliva is in accord with a priori expectation. These organisms are benign nonpathogens completely devoid of the ability to invade tissues; furthermore, the tissue they damage is immunologically inert. Rizzo,s2 using a very sensitive test system, showed that a soluble antigen was unable to permeate into healthy gingival tissue, and other workers fed L. acidophilus to volunteers for a period of 14 months without producing a detectable antibody response.63 On the other hand, there is a report that the mere presence of bacteria on teeth stimulated serum antibody production,15 and the writings of other workers seem to imply that, because dental caries is a bacterial disease, an immune response must necessarily occur.7l X2>14*2oa31v32 However, the tissues which produce immune responses are incapable of reason. They do not produce antibodies when apparently desirable but only when they are exposed to a sufficient concentration of a foreign antigen. They then produce antibodies, regardless of whether they are beneficial, damaging, or even lethal. 58 Production of antibodies to the vast number of antigens which pass through and are normally present in the mouth would be absurdly wasteful, and the low levels of antibody in saliva is proof enough that this does not occur. Those antigens to which immune responses are desirable will select themselves by penetrating the initial anatomic defenses of the oral cavity and reaching the immunologically reactive cells. Of course, harmless bacteria may be passively carried through breaches in the oral mucosa, may reach the dental pulp, or may have antigens which, though benign, are present on pathogenic bacteria. It is noteworthy that in this work salivary antibodies were detected only with respect to Str. pyogenes, the only pathogen among the bacteria tested.

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If effective concentrations of antibody specific for bacteria in the normal oral flora were present in saliva, then the studies of dental plaque using the direct fluorescent antibody technique64-66 could not have succeeded, for the salivary antibodies would have blocked the reaction of the specific fluorescent antibodies. As a final incongruity, there are the reports that caries-free subjects have higher levels of serum antibody reactive with lactobacilli and cariogenic streptococci than do subjects with many carious teeth. Thus, persons who have not suffered from the disease and have received the least antigenic stimulus paradoxically produce the greatest antibody response. Unfortunately, such inexplicable contradictions of the fundamentals of immunology are not rare in the field of caries immunity. The finding that cariogenic bacteria can be coated with antibody without producing any detectable effect on their rates of acid production will occasion little surprise. Antibodies neutralize toxins and facilitate phagocytosis and lysis by complement, but the simple union of antibody with a bacterium has not been shown to damage the organism or affect its metabolism.57 Mice rendered immune to pneumococcal infection by the stimulation of high levels of serum antibody succumb to the small numbers of pneumococci required to kill nonimmune mice if their phagocytic cells are depleted. 6T Only gram-negative bacteria have been shown to be lysed by complement or rendered susceptible to lysozyme by antibody. Furthermore, the elements of complement are not present in saliva although, paradoxically, immunoconglutinin is.6s Complement and lysozyme were added to the tests to check that the gram-positive cariogenic bacteria did not provide an exception to the rule, but the negative results were as anticipated. The massive uptake of cariogenic bacteria by rabbit polymorphs in the cornplete absence of serum was most impressive. Although specific antibodies did effect a marginal increase in phagocytosis, they were clearly of minor importance. These findings are at variance with those of Hammond and Weinmann69170 who found that serum opsonin greatly increased the phagocytosis of L. a&dophilus and an oral streptococcus. The technique used by these investigators and that used in the present work were so different that comparisons are precluded. If the results are applicable to oral polymorphs, then intact cells should be able to ingest and destroy cariogenic bacteria without difficulty. The presence of antibody might slightly aid the process, but the possibility of antibodies facilitating phagocytosis by defunct polymorphs in the unfavorable environment of the dental plaque can be absolutely discounted. The general assumption that opsonin, or antibody, is essential for phagocytosis arises from the fact that practically all the research on phagocytosis has been carried out with pathogenic bacteria. There has been little interest in the phagocytosis of harmless bacteria, such as those which cause dental caries. Even Hirsch and Strauss,61 who showed that complete phagocytosis could occur in the absence of serum, used nonpathogenic variants of the pneumococcus and Str. pyogenes as test organisms. Cariogenic bacteria appear to be as easily engulfed by polymorphs as are inert particles, and one is tempted to suggest that the distinction between a harmless commensal and a potential pathogen is that the former can be taken up by polymorphs without the need of serum factors. A thorough, but not exhaustive, search

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of the literature on immunoglobulin deficiencies failed to reveal a single mention of persons with low antibody levels being unusually prone to dental caries or other oral diseases. On the other hand, even slight degrees of agranulocytosis frequently produce clinical manifestations in the mouth. This matter of phagocytosis of cariogenic bacteria is a further illustration of the error and confusion that can result if it is assumed, without evidence, that facts established in studies of the common bacterial infections are applicable to a unique disease such as dental caries. The idea that antibodies may be protective in dental caries is simply speculation based on the results of some human and animal studies. Most of the human studies make the error, discussed in the first part of this article, of assuming that absence of cavities is synonymous with immunity to dental caries. They assert that caries-free persons have higher levels of serum antibody specific for cariogenic bacteria than do subjects with many carious lesions, but neither the way in which these high antibody levels were stimulated nor the mechanisms by which they protect from dental caries is explained. When lactobacilli were thought to be the cause of dental caries, groups of caries-free subjects had high levels of antibody reactive with lactobacilli; nowadays, they have high levels of antibody specific for dextran producing streptococci. Groups of cariesfree persons, it seems, have a remarkable propensity for producing high levels of antibody specific for whatever organism happens to be currently in vogue as the cause of dental caries! Animal studies purporting to show that immune responses are protective in dental caries are outnumbered by those in which no such effect could be demonstrated. Again, those who claim a protective role for antibodies fail to explain the mechanism of this protection; they simply infer that, because antibodies are known to be of value in other bacterial diseases, they must necessarily be effective in dental caries. It was to explore the possibility that antibodies might be of value in dental caries that the experiments reported in this article were carried out. Negative results are always disappointing but, in summary, they show that if antibodies specific for cariogenic bacteria are present in saliva at all, they are in very low concentration and, even if the amounts of antibody were greatly increased, there is no evidence that they would in any way modify the progress of dental caries. I wish to thank Mr. J. E. Tuyau for his technical assistance throughout the diagram, I am indebted to Miss Jennifer A. Middleton.

this work. For

REFERENCES

1. Carlsson, J.: Plaque Formation and Streptococcal Colonisation on Teeth, Odontol. Revy 19: Supp. 14, pp. 3-14, 1968. 2. Edwardsson, 8.: Characteristics of Caries-Inducing Human Streptococci Resembling Streptooocc~s mutans, Arch. Oral. Biol. 13: 637-646, 1968. 3. Edwardsson, 8.: The Caries-Inducing Property of Variants of Streptococcus mtitans, Odontol. Bevy 21: 153-157, 1970. 4. Guggenheim, B.: Streptococci of Dental Plaques, Caries Res. 2: 147-163, 1968. 5. Keyes, P. H.: Research in Dental Caries, J. Am. Dent. Assoc. 76: 1357-1373, 1968. 6. Burnett, G. W., and Scherp, H. W.: Oral Microbiology and Infectious Disease, ed. 3, Baltimore, 1968, Williams & Wilkins Company! pp. 352-358. 7. Jay, P., Crowley, M., Hadley, F. P., and Bunting, R. W.: Bacteriologic and Immunologic Studies of Dental Caries, J. Am. Dent. Assoc. 20: 2130-2148, 1933. 8. Canby, C. P., and Bemier, J. L.: Bacteriological and Immunological Studies in Dental Caries, J. Am. Dent. Assoc. 29: 606-617, 1942.

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9. Fitzgerald, R. J., and Keyes, P. H.: Attempted Immunization of Albino Hamsters Against Induced Dental Caries, Program and Abstracts, International Association for Dental Research, Abstr. 146, 1962. 10. Sweenev. E. A.. Shaw. J. H.. and Childs. E. L.: Effect of Passive Immunization on the Dental “Caries incidence of ‘Caries-Susceptible Rats, J. Dent. Res. 46: 993-997, 1966. 11. Wagner, M.: Specific Immunization Against Streptococcus faecalis-Induced Caries in the Gnotobiotic Rat, Bacterial. Proc., Abstr. 99, 1967. 12. Lehner, T., Cardwell, J. E., and Clarry, E. D.: Immunoglobulins in Saliva and Serum in Dental Caries, Lancet 1: 1294-1297, 1967. A. N., Pinter, J. K., Quillman, P. D., and Hayashi, J. A.: Immunization With 13. Bahn, Enzymes Against Caries in the Rat, Program and Abstracts, International Association for Dental Research. Abstr. 64. 1969. 14. Borkowski, J. M., Hayashi, J. A., Shklair, I. L., and Bahn, A. N.: Bactericidal Antibody to Caries-Inducing Streptococci, Program and Abstracts, International Association for Dental Research, Abstr. 65, 1969. 15. Berkenbilt. D. A.. Matsutani. K. K.. and Bahn. A. N.: Chronoloav of Develoument of Antibody to Cariogenic Bacteria, Program and Abstracts, Int&ational Association for Dental Research, Abstr. 312, 1969. 16. Bowen, W. H.: A Vaccine Against Dental Caries: A Pilot Experiment in Monkeys (Mamma irzLs), Br. Dent. J. 126: 159-160, 1969. 17. Gaffar, A., Marcussen, H. W., Ruffner, J., and Kestenbaum, R. C.: Effects of Specific Immunization on Experimental Dental Caries in Hamsters, Program and Abstracts, International Association for Dental Research, Abstr. 304, 1970. 18. Tanzer, J. M., Hageage, G. J., and Larson, R. H.: Inability to Immunologically Protect Rats Against Smooth Surface Caries, Program and Abstracts, International Association for Dental Research, Abstr. 466, 1970. 19. Genco. R. J.. Evans. R. T.. and Catlin. J.: Inhibition of In Vitro Plaaue Formed bv Cariogenic Streptococci, Program and Abstracts, International AssociaGon for Dental Research, Abstr. 625, 1970. T., Wilton, J. M. A., and Ward, R. G.: Serum Antibodies in Dental Caries in 20. Lehner, Man, Arch: Oral Biol. 15: 4811490, 1970. ’ 21. Guggenheim, B., Muhlemann, H. R., Regolati, B., and Schmid, R.: The Effect of Immunization Against Streptococci or Glucosyl-Transferases on Plaque Formation and Dental Caries in Rats. In McHugh, W. D. (editor) : Dental Plaque, Edinburgh, 1970, E. & S. Livingstone, Ltd., pp. 287-296. 22. Schiott, C. R., and Me, H.: The Origin and Variation in Number of Leucocytes in the Human Saliva, J. Periodontol. Res. 5: 36-41, 1970. 23. Claman, H. N., Merrill, D. A., and Hartley, T. F.: Salivary Immunoglobulins: Normal Adult Values and Dissociation Between Serum and Salivary Levels, J. Allergy 40: 1X-159, 1967. 24. Kraus, F. W., and Konno, J.: Antibodies in Saliva, Ann. N. Y. Acad. Sci. 106: 311-329. 1962. ’ ’ 25. Kraus, F. W., and Konno, J.: The Salivary Secretion of Antibody, Ala. J. Med. Sci. 2: 15-22. 1965. 26. Brandtzaeg, P.: Immunochemical Comparison of Proteins in Human Gingival Pocket Fluid, Serum and Saliva, Arch. Oral Biol. 10: 795-803,1965. J. C., Merrill, D. A., and Claman, H. N.: Salivary Immunoglobulin and Albumin: 27. Selner, Development During the Newborn Period, J. Pediatr. 72: 685-689, 1968. 28. Mandel, I. D., and Khurana, H. S.: The Relation of Human Salivary uA Globulin and Albumin to Flow Rate, Arch. Oral Biol. 14: 1433-1435,1969. 29. Oppenheim, F. G.: Preliminary Observations on the Presence and Origin of Serum Albumin in Human Saliva, Helv. Odontol. Acta 14: 10-17, 1970. 30. Brill, N.: The Gingival Pocket Fluid; Studies of Its Occurrence, Composition and Effect, Acta Odontol. Stand. 20: Supp. 32, l-115, 1962. J. F.: Immunoglobulin Formation and Function in Different Tissues, Curr. 31. Heremans, Top. Microbial., Immunol. 46: 131-203, 1968. 32. Tomaai, T. B., and Bienenstock, J.: Secretory Immunoglobulins, Adv. Immunol. 9: l-96, 1968. 33. Hobbs, J. R.: Immune Globulins in Some Diseases, Br. J. Hosp. Med. 3: 669-680, 1970. 34. Chauncey, H. H., Feller, R. P., Halsband, E. R., and Jaskiel, L. D.: Hemsgglutinine of Human Parotid and Submandibular Secretions, Proc. Sot. Exp. Biol. Med. 121: 130133. 1966. 35. Haworth, J. C., and Dilling, L.: Concentration of d-globulin in Serum, Saliva, and Nasopharyngeal Secretions of Infants and Children, J. Lab. Clin. Med. 67: 922-933, 1966. 36. South, M. A., Warwick, W. J., Wollheim, F. A., and Good, R. A.: The IgA System. IlI. IgA Levels in the Serum and Saliva of Pediatric Patients-Evidence for a Local Immunological System, J. Pediatr. 71: 645-653, 1967.

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37. Genco, R. J., and Taubman, M. A.: Secretory aA Antibodies Induced by Local Immunization, Nature 221: 679-681, 1969. 38. Tomasi, T. B.: On the Mechamsms of Transport and Biological Significance of Antibodies in External Secretions, Arthritis Rheum. 12: 45-50, 1969. 39. Smith, R. T.: Gamma-A Immunoglobulins and the Concept of Local Immunity, Pediatrics 43: 317-320, 1969. 40. Mancini, G., Carbonara, A. O., and Heremans, J. F.: Immunochemical Quantitation of Antigens by Single Radial Immunodiffusion, Immunochemistry 2: 235-246, 1965. 41. Evans, R. T., and Mergenhagen, S. E.: Occurrence of Natural Antibacterial Antibody in Human Parotid Fluid, Proc. Sot. Exp. Biol. Med. 119: 815819, 1965. 42. Douglas, R. G., Rossen, R. D., Butler, W. T., and Couch, R. B.: Rhinovirus Neutralizing Antibodv in Tears. Parotid Saliva, Nasal Secretions and Serum, J. Immunol. 99: 297303, 196i. 43. Tourville, D., Bienenstock, J., and Tomasi, T. B.: Natural Antibodies of Human Serum, Saliva and Urine Reactive With Escherichia coli, Proc. Sot. Exp. Biol. Med. 128: 722727, 1968. 44. Sirisinha, S.: Reactions of Human Salivary Immunoglobulins With Indigenous Bacteria, Arch. Oral Biol. 15: 551-554, 1970. 45. Williams, N. B.: Antigenic Components of Lactobacilli of Human Oral Origin, J. Infect. Dia. 82: 31-41, 1948. 46. Sharue. M. E.: A Seroloeical Classification of Lactobacilli. I J. Gen. Microbial. 12: w 107-l-22, 1955. 47. Bratthall, D.: Immunodiffusion Studies on the Serological Specificity of Streptococci Resembling Strewtococcus mutans. Odontol. Rew 20: 231-244, 1969. 48. Bratthall, “D.: Demonstration of Five Serological Groups of Streptococcal Strains Resembling Streptococcus nratans, Odontol. Revy 21: 143-152, 1970. 49. Carlsson, J.: Presence of Various Types of Non-haemolytic Streptococci in Dental Plaque and in Other Sites of the Oral Cavity in Man, Odontol. Revy 18: 55-74, 1967. J. M.: Human Oral Leukocytes, Periodontics 1: 109-117, 1963. 50. Klinkhamer, 51. Rovelstad, G. H.: Salivary Corpuscle Activity, J. Am. Dent. Assoc. 68: 364-373, 1964. 52. Sharry, J. J., and Krasse, B.: Observations on the Origin of Salivary Leukocytes, Acta Odontol. &and. 18: 347-358. 1960. 53. Wright, D. E.: The Source and Rate of Entry of Leucocytes in the Human Mouth, Arch. Oral Biol. 9: 321-329, 1964. 54. Attstrom, R.: Presence of Leucocvtes in Crevices of Healthv and Chronically Inflamed Gingivae; J. Periodontol. Res. 5: 42-47, 1970. 55. Wright, D. E., and Jenkins, G. N.: Leucocytes in the Saliva of Caries-Free and CariesActive Subjects, J. Dent. Res. 32: 511-523, 1953. 56. Florey, H. W., and Jennings, M. A.: Chemotaxis, Phagocytosis and the Formation of Abscesses. In Florey, H. W. (editor): The Reticula-Endothelial System in General Pathology, ed. 4, London, 1970, Lloyd-Luke, Ltd., pp. 124-174. and 57. Wilson, G. S., and Miles, A. A.: Topley and Wilson’s Principles of Bacteriology Immunity, ed. 5, London, 1964, Edward Arnold, Ltd., vol. 2, pp. 1291-1292. 58. Humphrey, J. H., and White, R. G.: Immunology for Students of Medicine, ed. 3, Oxford, 1970, Blackwell Scientific Publications, pp. 52 and 170. 59. Bladen, H., Hageage, G., Harr, R., Pollock, F., and Scott, S.: Lysozyme Sensitivity of Certain Oral Bacteria After Treatment With Complement, Program and Abstracts, International Association for Dental Research, Abstr. 154, 1970. 60. Fitzgerald, R. J., and Jordan, H. V.: Polysaccharjde Producing Bacteria and Caries. In Harris, R. 8. (editor) : Art and Science of Dental Caries Research, New York, 1968, Academic Press, Inc., pp. 79-87. 61. Hirsch, J. G., and Strauss, B.: Studies on Heat-Labile Opsonin in Rabbit Serum, J. Immunol. 92: 145-154, 1964. 62. Rizzo, A. A.: Histologic and Immunologic Evaluation of Antigen Penetration Into Oral Tissues After Tooieal ADDlication. J. Periodontol. 41: 210-213. 1970. 63. Gillespie, B. H., Steb&, M., Sgott! E. N., and Christ, Y. 8.: Serological Relationships Existing Between Bacterial Parasites and Their Hosts. 1. Antibodies in Human Blood Serum for Native Intestinal Bacteria, J. Immunol. 65: 105-113, 1950. 64. Ritz, H. L.: Localization of Nocardia in Dental Plaque by Immunofluorescence, Proc. Sot. Exp. Biol. Med. 113: 925-929, 1963. 65. Snyder, M. L., Bullock,. W. W., and Parker, R. B.: Morphology of Gram-Positive Filamentous Bacteria Identified in Dental Plaque by Fluorescent Antibody Technique, Arch. Oral Biol. 12: 1269-1273, 1967. 66. Ritz, H. L.: Fluorescent Antibody Staining of Neisseria, Streptococcus and Veillonella in Frozen Sections of Human Dental Plaque, Arch. Oral Biol. 14: 1073-1088, 1969. 67. Rich, A. R., and McKee, C. M.: A Study of the Character and Degree of Protection Afforded by the Immune State Independently of the Leucocytes, Bull. Johns Hopkins Hosp. 54: 277-311, 1934.

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68. Lachmsnn, P. J., and Thomson, R. A.: Immunoconglutinins in Human Saliva-A Group of Unusual IgA Antibodies, Immunolo y 18: 157-169, 1970. 69. Hammond, C. W., and Wemmann, J. !S.: Opsonin in Saliva, J. Dent. Res. 21: 279-233, 1942. 70. Hammond, C. W., and Weinmann, J. P.: Opsonin in the Saliva of Caries-Negative and Caries-Positive Patients, J. Dent. Res. 21: 509-511, 1942. 12eprbt

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Dr. W. Sims Pathology Department Royal Dental Hospital University of London L&ester Square London W.C.2, England